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Single Mouse Cells Producing Two Antibody Molecules and Giving Rise to Antigen-Driven Intraclonal Variation after Immunization with Two Unrelated Antigens¹

From the Institut d'Immuno-Biologie, (U. 20 INSERM, LA 143 CNRS), 96, rue Didot, 75674 Paris Cedex 14, France

Abstract

Hemolytic plaque-forming cells (PFC) simultaneously lysing two different hapten-red cell indicators (double PFC) appear regularly along with PFC lysing only one indicator, on the 4th to 5th day of secondary response in mice primed 2 to 4 months before with hapten-protein conjugates mixed in Freund's complete adjuvant. Addition of soluble-specific inhibitors, i.e., the same haptens conjugated to different protein-carriers, results in an inhibition of the corresponding specific lytic activity and the disappearance of the double PFC. Micromanipulation of these cells from the initial medium into a second medium, containing both indicator RBC and one specific inhibitor, inhibited the corresponding specific lysis in 80% of double cells without affecting the unrelated lytic activity. Subsequent micromanipulation into a third inhibitor-free medium resulted in a recovery of the double lysis in more than 50% of them. This evidence for the synthesis of two different antibody molecules by double PFC was further strengthened by individual cell culture experiments; most of these cells divided and engendered daughter PFC-secreting antibody of only one of the two specificities, thus showing first, the transient nature of double production, and second, that each lytic activity can be inherited separately.

Individual cell cultures also showed occurrence of clonal variation in progeny of single PFC. Among PFC from mice immunized with two antigens to progeny variation occurred in 24 out of 46 (0.656) parental cells as against only 1 out of 40 (0.025) observed among individual PFC from animals immunized with one antigen. Moreover, this variation was antigen driven since absence for 48 hr of the one antigen resulted in discontinuance of generation of daughter PFC of the corresponding specificity. A similar degree of antigen-driven variation was observed when spontaneous PFC or PFC arising early after immunization (2nd or 3rd day) with one antigen were individually cultured for 48 hr with a different antigen; one part of parental PFC (0.545) yielded variant daughters of the specificity of the latter antigen, whereas the other part (0.455) yielded progeny of the same as the parental PFC specificity in a clonal manner. These results suggest that differentiation of B cells during the early phase after antigenic stimulation would involve a process of intracellular selection and/or rearrangement of V genes.

Footnotes

¹ This work was supported by Grants CRL-77 5 070, INSERM and ATP 3615 CNRS.

² To whom correspondance should be addressed.